Project Summary The germline produces gametes that transmit parental epigenetic and genetic information to offspring. The maintenance of germline and the development of functional gametes require Piwi, an ancient member of the Argonaute family, and Piwi-interacting small RNAs (piRNA). Compared to other types of small RNAs (e.g., microRNAs and siRNAs), piRNA biogenesis remains mysterious largely owing to their exclusive expression in the germline and sequence diversity between organisms. Over the past decade, significant progress has been made toward elucidating the function of the piRNA pathway as a defense mechanism against transposable elements. Yet organisms including worms and mice express thousands of piRNAs that do not correspond to transposon sequences, suggesting that piRNAs may have alternative functions. My preliminary findings suggest that C. elegans piRNAs regulate endogenous gene expression and influence developmental processes. The goal of this proposal is to address fundamental questions regarding the piRNA pathway: How is piRNA expression controlled at the transcription level? How are piRNA precursors processed into mature piRNAs? Do piRNAs regulate the expression of germline genes? And how do they influence cellular and developmental processes? During the K99 phase of this award, I will: (1) define the transcription program of piRNA producing loci and chromatin factors associated with piRNA genes; (2) characterize a cap structure on piRNA precursors and study its role in piRNA expression; (3) established a cell free system to characterize enzymatic activity of Piwi and piRNA-processing enzymes. During the R00 phase of the award, I will: (1) continue the in vitro biochemical studies; (2) determine how piRNAs regulate developmental processes such as dosage compensation; (3) investigate potential auto-regulation of Piwi expression. Taken together, the proposed research will provide a framework for studying piRNA biogenesis in C. elegans and in other species. It will greatly advance our understanding of mechanism and functions of piRNAs in germline maintenance and gametogenesis. Ultimately the work will provide new tools and therapies that will improve human fertility and health.